Modulation of intestinal immune response by Outer Membrane Vesicles from probiotic and commensal Escherichia coli strains

Gut microbiota play a critical role in maintaining human intestinal homeostasis and host health. Bacterial extracellular vesicles are key players in bacteria-host communication as they allow delivery of effector molecules into the host cells. The aim of this thesis was to evaluate the immunomodulatory activity of extracellular vesicles (OMVs) released by two microbiota strains, the probiotic strains Escherichia coli Nissle 1917 (EcN) and the commensal ECOR12. Results obtained using in vitro and ex vivo models of intestinal barrier demonstrated that EcN and ECOR12 OMVs were able to be internalized in the Caco-2 cell line and also immunomodulate through the intestinal epithelium. In addition, OMVs released by Gram-negative bacteria can carry many ligands of PPRs, which are key components of innate immunity, and we have demonstrated that part of the immune responses triggered after EcN and ECOR12 OMVs stimulations were mediated by the activation of NOD1-signalling pathways at early endosomal level by bacterial peptidoglycan contained within OMVs. On the other hand, using an in vivo model of colitis in mice induced by dextran sodium sulphate (DSS), we confirmed the anti- inflammatory properties and the beneficial effect on the intestinal barrier exerted by EcN OMVs (5 µg/day), that were able to counteract most of the inflammatory damages caused by the DSS intake. Finally, we also tested the effect of secreted factors released by EcN on Salmonella enterica serovar Typhymurium infection, two pathogenic bacteria with high impact on health society. Results showed that E. coli supernatants from both EcN and other commensal E. coli strains, but not OMVs reduce Salmonella invasion. Enzymatic treatment confirmed that the active secreted factor is not a protein, DNA or RNA molecules. We also observed that EcN supernatant slightly reduces secretion of invasion Salmonella proteins, and these results suggests that probably EcN secreted factors might diminish Salmonella invasion by interfering with the T3SS secretion system. In conclusion, data presented in this thesis work show that OMVs released by microbiota E. coli strains have a key role in modulating host immune responses and they have a key role to maintain the intestinal homeostasis